Spatial immune heterogeneity of hypoxia-induced exhausted features in high-grade glioma

The tumor immune microenvironment (TIME) in high-grade glioma (HGG) exhibits high spatial heterogeneity. Though the tumor core and peripheral regions have different biological features, the cause of this spatial heterogeneity has not been clearly elucidated. Here, we examined the spatial heterogeneity of HGG using core and peripheral regions obtained separately from the patients with HGG. We analyzed infiltrating immune cells by flow cytometry from 34 patients with HGG and the transcriptomes by RNA-seq analysis from 18 patients with HGG. Peripheral region-infiltrating immune cells were in vitro cultured in hypoxic conditions and their immunophenotypes analyzed. We analyzed whether the frequencies of exhausted CD8⁺ T cells and immunosuppressive cells in the core or peripheral regions are associated with the survival of patients with HGG. We found that terminally exhausted CD8⁺ T cells and immunosuppressive cells, including regulatory T (T_REG) cells and M2 tumor-associated macrophages (TAMs), are more enriched in the core regions than the peripheral regions. Terminally exhausted and immunosuppressive profiles in the core region significantly correlated with the hypoxia signature, which was enriched in the core region. Importantly, in vitro culture of peripheral region-infiltrating immune cells in hypoxic conditions resulted in an increase in terminally exhausted CD8⁺ T cells, CTLA-4⁺ T_REG cells, and M2 TAMs. Finally, we found that a high frequency of PD-1⁺CTLA-4⁺CD8⁺ T cells in the core regions was significantly associated with decreased progression-free survival of patients with HGG. The hypoxic condition in the core region of HGG directly induces an immunosuppressive TIME, which is associated with patient survival.